Posttranslational Regulation of Human DNA Polymerase ι*

Background: Many proteins are subject to posttranslational regulation, such as ubiquitination. Results: Human DNA polymerase ι (polι) can be monoubiquitinated at >27 unique sites, and exposure to naphthoquinones results in polyubiquitination of polι. Conclusion: Ubiquitination sites are located across the entire polι polypeptide as well as various structural motifs. Significance: Ubiquitination at these sites is likely to alter cellular functions of polι in vivo.

when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, pol becomes transiently polyubiquitinated via Lys 11 -and Lys 48 -linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to pol occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys 11 and Lys 48 rather than oxidative damage per se.
To survive the constant threat to their genomes from exposure to endogenous and exogenous DNA-damaging agents, cells are equipped with an impressive array of DNA repair mechanisms. However, situations arise where DNA lesions in the genome remain unrepaired and cells are forced to tolerate the DNA damage. One such tolerance mechanism is "translesion DNA synthesis" (TLS). 2 During TLS, the high fidelity replicase, which is unable to traverse the DNA lesion due to its constrained active site, is replaced with a specialized DNA polymerase (pol) with a more spacious active site that can accommodate the damaged DNA (1). Many of the DNA polymerases discovered in the past 15 years appear to have some capacity to promote TLS. However, the best characterized TLS polymerases belong to the Y-family of DNA polymerases (2). Y-family DNA polymerases are typified by human pol, which bypasses a thymine-thymine cyclobutane pyrimidine dimer efficiently and with much higher accuracy than any other human TLS polymerases (3). Because of their more spacious active sites (4), the TLS enzymes are also able to accommodate non-canonical Watson-Crick base pairing and are usually much more errorprone than high fidelity replicases when they replicate undamaged DNA (1). In specialized situations, such as during immunoglobulin somatic hypermutation, this creates genetic diversity and leads to high affinity antigen-specific immunoglobulins (5). However, under normal circumstances, random mutagenesis of chromosomal DNA is highly deleterious, often leading to mutagenesis and tumorigenesis in higher organisms.
It is clear, therefore, that the activity of the TLS polymerases needs to be tightly regulated so that they only gain access to undamaged genomic DNA when appropriate. Previous studies have revealed that the posttranslational modification of the TLS polymerases themselves or their interacting partners plays a major role in regulating their cellular activities (for a review, see Ref. 6). In particular, both mono-and polyubiquitination appear to play a central role in regulating TLS polymerases because monoubiquitination of the proliferating cell nuclear antigen (PCNA) appears to control the switch between high fidelity replicases and TLS polymerases (7,8). However, PCNA can be further polyubiquitinated. The addition of Lys 63 -linked polyubiquitin chains to PCNA that is monoubiquitinated at Lys 164 leads to a damage avoidance template-switching pathway that in contrast to TLS allows for error-free DNA damage bypass (9,10).
In addition to PCNA, both human pol and pol TLS polymerases are also subject to monoubiquitination (11,12). Attaching a single ubiquitin moiety to one of four lysine residues in the C terminus of pol blocks the physical interaction between pol and PCNA (12). As a consequence, pol needs to be actively deubiquitinated prior to interacting with PCNA and subsequently recruited to a stalled replication fork (12). The cellular role of pol monoubiquitination remains enigmatic. However, our previous results suggest that monoubiquitination of either pol or pol is a prerequisite for the physical and functional interaction between the two polymerases (13).
Human pol is one of the least accurate DNA polymerases and exhibits a 10,000-fold range in base substitution fidelity depending on the template sequence copied (for a review, see Ref. 1). Pol has been extensively characterized at the biochemical level (14 -19), and its in vivo relocalization in response to DNA damage has been shown (20,21). The enzyme is involved in the error-free bypass of methylglyoxal-induced minor groove lesions, N 2 -carboxyethyl-2Ј-deoxyguanosine (22), and a deficiency in pol has been suggested to cause sensitivity to oxidative and 4-hydroxynonenal DNA damage (23,24) as well as stimulate UV-induced mesenchymal carcinogenesis (25). However, the primary biological function of pol is still far from being understood.
Some assumptions on the cellular role of pol can be derived from the various domains/motifs identified in pol. The Nterminal part of the protein contains two partly overlapping catalytic domains, a DNA polymerase domain and a 5Ј-deoxyribose phosphate lyase domain (26,27). The core polymerase domain is built of palm, finger, thumb, and little finger subdomains (28,29). The C-terminal portion of the protein is unstructured and devoted to facilitating interactions with a variety of proteins. Similar to other TLS polymerases, pol contains a PCNA-interacting peptide motif responsible for the interaction with PCNA (30 -32) and a Rev1-interacting region (33,34). It also contains two ubiquitin binding motifs (11).
Typically, the conjugation of ubiquitin to the lysine residue of a substrate protein occurs as a result of a three-enzyme cascade process involving ubiquitin-activating enzyme (E1), ubiquitinconjugating enzyme (E2), and ubiquitin ligase (E3) (35). Ubiquitin contains seven lysine residues (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 , and Lys 63 ) and is itself a target for further ubiquitination. Indeed, repetitive ubiquitination can establish polyubiquitin chains on a target protein. The length, type of linkage, and consequent shape of conjugated polyubiquitin chains direct the function and processing of many intracellular proteins in eukaryotes (for a review, see Ref. 36). All types of ubiquitin chains exist in the cell; however, they vary in abundance and functionality. Different types of polyubiquitin chains regulate different biological processes by promoting proteasomal degradation, altering subcellular localization, modulating enzymatic activity, and facilitating protein-protein interactions (37).
In the current work, we used mass spectrometry analysis to identify the lysine residues that can be ubiquitinated in human pol. In contrast to PCNA, which is primarily ubiquitinated at Lys 164 , and pol where the ubiquitinated residues are clustered in its C terminus, the monoubiquitinated residues in pol are scattered among its various functional domains/motifs. Furthermore, unlike monoubiquitinated pol that is deubiquitinated upon UV irradiation, the level of monoubiquitinated pol remains unchanged after exposure to UV irradiation, ethyl methanesulfonate, mitomycin C, or potassium bromate. Interestingly, however, after exposure to menadione and structurally related naphthoquinones, pol is rapidly polyubiquitinated, and intracellular levels of both the unmodified and the monoubiquitinated forms of pol decrease significantly. We present evidence that the polyubiquitination of pol is not in response to oxidative DNA damage per se but is rather due to the inhibition of KAT3B/p300-dependent acetylation of ubiquitin, which in turn allows for the formation of Lys 11 -and Lys 48 -linked polyubiquitin chains on pol that subsequently target it for degradation.
Mammalian Expression Plasmids-Plasmid pJRM46 is a derivative of pCMV6AN-DDK (Origene Technologies, Rockville, MD), which expresses N-terminal FLAG-tagged fulllength human pol (13). Derivatives with single or multiple Lys 3 Ala or Lys 3 Arg substitutions were generated by chemically synthesizing appropriate DNA fragments (Genscript) that were subsequently cloned into pJRM46. Plasmid pRK7-POLI-3XFLAG is a derivative of pRK7 (38), which expresses full-length human pol with three C-terminal FLAG tags. The vector was constructed by inserting three tandem repeats of the FLAG epitope tag (DYKDDDDK) into the BamHI and EcoRI sites of pRK7 to generate pRK7-3XFLAG. The full-length human POLI gene was amplified from HEK293T cells by reverse transcription-PCR using primers POLI-S (AAAGCTA-GCATGGAGAAGCTGGGGGTGGA) and POLI-AS (AAAG-GATCCTTTATGTCCAATGTGGAAATCT). These primers introduce 5Ј NheI and 3Ј BamHI sites into the amplicon, which was subcloned into the XbaI and BamHI sites of the pRK7-3-XFLAG vector. A full list of plasmids used in the current study is shown in Table 1.
Plasmid Transfection, Protein Expression, and Western Blotting-HEK293T cells were plated onto 100-mm culture plates at a seeding density of 3 ϫ 10 6 cells. When cells were ϳ40% confluent, plasmids were transfected into cells using Turbofectin 8.0 according to the manufacturer's instructions (Origene Technologies). Cells were either mock treated or exposed to a variety of agents 24 or 48 h after transfection depending upon the treatment times required. At appropriate times thereafter, cells were gently collected, washed twice with cold Dulbecco's PBS without calcium or magnesium, suspended in modified radioimmunoprecipitation assay buffer (RIPA buffer) (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, 1 mM Na 3 VO 4 , and Sigma protease inhibitor mixture), and lysed by sonication for 10 s. Immediately after sonication, the extracts were clarified by centrifugation at ϩ4°C for 15 min in a Sorvall Biofuge Pico at 16,000 ϫ g. The supernatants (extracts) were transferred to fresh tubes, and protein concentrations were measured using the Pierce BCA Assay (Pierce Biotechnology). Cell extracts were kept at ϩ4°C until being separated by 4 -16% gradient SDS-PAGE. Proteins were transferred to a PVDF membrane, and FLAG-tagged pol was visualized using a Tropix Western-Star chemiluminescence kit using mouse anti-FLAG monoclo-nal antibodies (Abnova) followed by secondary anti-mouse antibody (Novagen). Where noted, the following other antibodies were used: POLI monoclonal antibody (M01) clone 8G9 (Abnova) or polyclonal rabbit antibodies raised against a keyhole limpet hemocyanin-conjugated peptide corresponding to the C-terminal 15 amino acid residues of pol (20). Where noted, the level of pol was compared with ␤-actin present in the extracts that was visualized using rabbit anti-␤-actin antibodies (Cell Signaling Technology). The intensity of the individual bands was quantified using the ImageJ 1.47 application (National Institutes of Health).
Mass Spectrometry Analysis-Purified recombinant N-terminal FLAG-tagged human pol was purchased from Origene Technologies and was supplied at a final concentration of 0.106 mg/ml. Roughly 80% of the total protein represents unmodified FLAG-pol, and ϳ10% represents a slower migrating modified FLAG-pol (Fig. 1A). A total of 5 g of the combined FLAG-pol preparation was applied to a 1-mm-thick precast 10% polyacrylamide NuPAGE gel (Life Technologies). Proteins were separated by SDS-PAGE by running the gel at 190 V for 3.75 h. All subsequent solutions were prepared using ultrapure HPLC water. The gel was lightly stained using the Novex Collodial Blue Staining kit (Life Technologies) in a brand new disposable plastic tray. Unmodified and modified FLAG-pol bands were excised using a brand new hard backed razor blade under water. Gel fragments were washed two times with 50% acetonitrile in ultrapure water. Samples were sent to the Harvard Microchemistry Department (Harvard University, Cambridge, MA) where they were analyzed by mass spectrometry as a custom contract service.
LC-MS/MS experiments were performed as described previously (39). Briefly, the peptides were separated on an EASY- The images clearly show that the major band observed in the Coomassie Brilliant Blue-stained gel corresponds to native FLAG-pol. The slower migrating band also contains pol and appears to be a posttranslationally modified form of pol. The faster migrating band observed in A does not appear to be related to FLAGpol because it does not cross-react to either the FLAG or pol antibodies. Ub, ubiquitin.

Results
Sites of Ubiquitination in Pol-It has been reported previously that pol is monoubiquitinated in vivo (11). However, at the time that we embarked on these studies, the location of the modified residue(s) had yet to be determined. To identify the site(s) of ubiquitination in pol, we initially utilized the contract services of the Harvard Microchemistry Department (Harvard University) to provide mass spectrometry analysis of a commercially available preparation of N-terminal FLAG-tagged human pol (Origene Technologies) (Fig. 1A). The preparation contained a significant amount of a slower migrating protein that cross-reacts with anti-FLAG antibodies as well as both polyclonal and monoclonal antibodies against pol (Fig. 1B). Based upon the earlier work of Bienko et al. (11), we hypothesized that the slower migrating band was likely to be monoubiquitinated pol.
Mass spectrometry analysis of the isolated slower migrating FLAG-pol protein indicated it was indeed monoubiquitinated pol, which was modified at six unique lysine residues (Lys 248 , Lys 522 , Lys 526 , Lys 530 , Lys 549 , and Lys 704 ). Although we had limited mass spectrometry coverage of the very C terminus of pol, we rationalized that the C-terminal Lys 715 residue might also be subject to ubiquitination because it is most probably localized on the surface of the protein and thus likely to be exposed to ubiquitinating enzymes.
To determine which of the residues might be the primary site of pol ubiquitination, we transfected human HEK293T cells with a series of recombinant plasmids carrying FLAG-tagged pol, each containing a single lysine to alanine substitution (K248A, K522A, K526A, K530A, K549A, K704A, and K715A), and checked the extent of pol ubiquitination by Western blotting and probing with anti-FLAG antibodies. Remarkably, most of the pol mutants were ubiquitinated at levels comparable with the wild-type protein ( Fig. 2A). Interestingly, the largest reduction in ubiquitination occurred in the K715A mutant, which exhibited ϳ60% of the level observed with the wild-type protein, indicating that Lys 715 is indeed a target for ubiquitination. Based on these observations, it appears that none of the seven lysines is an exclusive site of ubiquitination. However, we rationalized that modification at lysines in close proximity to the respective alanine substitution might mask the effect of individual lysine mutations (e.g. Lys 522 , Lys 526 , and Lys 530 ). To test this hypothesis, we determined the extent of ubiquitination of pol mutants containing multiple Lys 3 Ala substitutions.   (Fig. 2B). To our surprise, ubiquitination of pol in the septuple mutant was only diminished by ϳ50% compared with the wild-type protein, suggesting the existence of additional ubiquitination sites in pol. By analogy to pol where a K682A substitution leads to ubiquitination at nearby lysine residues (12), we made Lys 3 Ala substitutions at pol residues Lys 237 , Lys 245 , and Lys 550 . Based on the results obtained by Wagner et al. (40), who reported proteome-wide analysis of in vivo ubiquitination sites, we also made Lys 3 Ala substitutions at Lys 267 and Lys 271 . However, a duodecuple mutant carrying all 12 Lys 3 Ala sub-stitutions did not prevent monoubiquitination of pol (Fig. 2B), thereby implying additional sites of ubiquitination in pol.
We therefore undertook an independent mass spectrometry analysis approach, this time using C-terminal FLAG-tagged pol. Interestingly, we identified six ubiquitination sites in pol that were clustered in the N-terminal half of the polymerase (Lys 53 , Lys 283 , Lys 309 , Lys 271 , Lys 310 , and Lys 320 ). None of these sites emerged in the original analysis of N-terminal FLAGtagged pol performed at the Harvard Microchemistry Department, and only one residue (Lys 271 ) was identified in the earlier studies by Wagner et al. (40).
Thus, by three independent approaches, two specifically focused on pol (described herein) and one proteome-wide Lysine residues in pol that have been shown to be subject to ubiquitination are indicated with a colored dot. The reference for each residue is given below the primary amino acid sequence of pol. As noted, pol can be ubiquitinated at more than 27 unique lysine residues. Some residues have been observed in multiple studies, but no single residue has been identified in every study. The various motifs and domains in pol are identified by color-coded bars above the primary amino acid sequence. dRP, 5Ј-deoxyribose phosphate; PIP, PCNA-interacting peptide motif. (Wagner et al. (40)), 17 independent ubiquitination sites in pol were identified. We were interested in determining whether substitutions at these sites would finally block ubiquitination of pol. Given the close structural proximity of pol Lys 51 and Lys 72 residues to Lys 53 (29), we also made substitutions at these residues. The combined mutant has 19 Lys 3 Ala substitutions. We noted that this mutant has altered gel electrophoretic mobility (Fig. 3A) and decided to limit the influence of the multiple alanine substitutions on the global charge of the protein and consequently its structure, so we also generated a 19-Lys 3 Arg mutant.
Interestingly, the 19-Lys3 Ala mutant showed 60% higher levels of pol ubiquitination than the 19-Lys 3 Arg mutant (Fig.  3), suggesting that multiple Lys 3 Ala mutations probably changed the structure of pol and possibly exposed lysine residues that perhaps would not normally be subject to monoubiquitination. We also observed the same effect when comparing the 12-Lys 3 Ala with the 12-Lys 3 Arg mutants. In light of the fact that the Lys 3 Arg changes in the combined pol mutants reduced ubiquitination of pol more acutely than the Lys 3 Ala mutations, we decided to re-evaluate the effect of a single K715R substitution because the K715A mutant gave the greatest reduction in the levels of ubiquitination (Fig. 2). Again, the conservative K715R substitution diminished ubiquitination of pol to a greater extent than K715A. In some regards, this is surprising because one might expect that the extreme C-terminal residue would be exposed and there would not be a large structural effect of the alanine or arginine substitutions.
Recent technical progress in mass spectrometry-based methods in combination with novel ubiquitin enrichment strategies using di-Gly-Lys-specific antibody (41) have significantly increased the number of documented ubiquitinated proteins and pinpointed many of their ubiquitin-modified lysines on a global level, including many TLS proteins (6). Within the last 4 years, several groups have reported large scale detection of lysine ubiquitination events in human cells, and in nine of these studies, ubiquitination sites of pol have been identified (41)(42)(43)(44)(45)(46)(47)(48)(49). Fig. 4 summarizes all the lysine residues in pol that have been shown to be ubiquitinated. In total, 27 lysine residues of pol have been experimentally shown to be ubiquitinated. Based upon structural considerations, we have identified another three lysine residues that could potentially be ubiquitinated. Eight of the sites were detected just once. The remaining 19 ubiquitination sites in pol were identified in anywhere between two and six independent studies, often using very different experimental strategies (e.g. ectopically expressed versus chromosomally expressed pol and/or different detection methods). However, no single site has been identified in all of the posttranslational modification studies. Thus, unlike pol, which is ubiquitinated at one primary site and a handful of secondary sites (12), pol does not appear to possess a primary site for ubiquitination but is instead ubiquitinated at multiple lysine residues.
Pol Ubiquitination in Response to a Variety of DNA-damaging Agents-The 30 potential ubiquitination sites are distributed along the entire length of the pol peptide. However, some are clustered in domains and motifs of pol that are important for its cellular function in DNA damage tolerance (5Ј-deoxyribose phosphate lyase domain, catalytic polymerase domain, PCNA-interacting peptide motif, Rev1-interacting region, UBM1, and UBM2) (Fig. 4). We were therefore interested in determining whether the ubiquitination status of pol is influenced by exposure to DNA-damaging agents. Indeed, there is a precedent for damage-induced deubiquitination of human pol to allow it to interact with ubiquitinated PCNA and facilitate TLS (12). We therefore examined pol ubiquitination in response to treatment with agents that cause different types of DNA damage such as UV irradiation, which results in both cyclobutane pyrimidine dimers and 6-4 photoproducts; ethyl methanesulfonate, which generates alkylation damage (50); mitomycin C, which generates interstrand cross-links (51,52); and two oxidizing agents, potassium bromate and menadione (53,54). Somewhat surprisingly, most of the DNA-damaging agents did not result in any significant change in the extent of pol ubiquitination even several hours after the initial treatment (Fig. 5, A-D). In contrast, in cells treated with 30 M menadione for 1 h (time 0), we observed an increase of pol with much slower mobility that is consistent with polyubiquitination of pol (Fig. 5E). Furthermore, the intracellular levels of pol decreased significantly 3-5 h after treatment, suggesting that the posttranslationally modified pol protein is targeted for degradation.

Pol Ubiquitination in Response to Treatment with Various Naphthoquinones and Inhibitors of Mitochondrial Function-
Our observation that potassium bromate did not elicit the same polyubiquitination of pol as menadione suggests that polyubiquitination of pol is unlikely to occur as a result of oxidative damage per se but occurs in specific response to menadione treatment. Although both agents cause oxidative DNA damage, they do so by different mechanisms. For example, potassium bromate induces glutathione-mediated oxidative base damage (53), whereas menadione does so by inducing mitochondrial dysfunction, leading to an increase in reactive oxygen species (54). However, we observed no significant increase in pol polyubiquitination after inhibition of the mitochondrial respiratory chain complex I with rotenone (55) or antimycin A, which inhibits cytochrome c reductase and the production of ATP (56) (Fig. 6), indicating that simple mitochondrial dysfunction is not the root cause for pol polyubiquitination. In contrast, we discovered that pol polyubiquitination occurs after exposure to naphthoquinones that are structurally related to menadione (Fig. 7), including 1,4-naphthoquinone, juglone, and plumbagin. Similar to menadione, all three compounds stimulated pol polyubiquitination in a concentrationdependent manner (Fig. 7). A particularly strong effect was observed after exposure to low concentrations of juglone and plumbagin (Fig. 7, C and D). However, another naphthoquinone, DMNQ, that also causes significant oxidative DNA damage (57) did not induce pol polyubiquitination (Fig. 8).
Although the UV irradiation-induced ubiquitination/deubiquitination of pol and pol seem to be differentially regulated (c.f. Ref. 12 and Fig. 5A), pol also appears to undergo polyubiquitination in response to menadione and plumbagin treatment. 3 Whether this occurs as a result of a common pathway controlling the polyubiquitination of both polymerases in response to naphthoquinones remains to be determined.
Pol Ubiquitination in Response to Treatment with an Inhibitor of KAT3B/p300 -It is apparent that the effects of menadione, juglone, 1,4-naphthoquinone, and plumbagin cannot be simply attributed to oxidative DNA damage or mitochondrial dysfunction. However, naphthoquinones are also known to inhibit the activity of the lysine acetyltransferase KAT3B/p300 (58,59). We therefore considered the possibility that the inhibition of ubiquitin acetylation may promote the polyubiquitination of pol. To test this hypothesis, HEK293T cells were treated with the KAT3B/p300 inhibitor L002. Indeed, similar to the effects of naphthoquinones, L002 resulted in the polyubiquitination of pol (Fig. 7E).
Mass Spectrometry Analysis of Polyubiquitinated Forms of Pol-To further explore the nature of polyubiquitinated forms of pol appearing after treatment with naphthoquinones, we performed mass spectrometry analysis on the purified ubiquitin-conjugated pol. To do so, we used anti-FLAG M2 beads (Sigma) to pull down N-terminal FLAG-tagged pol expressed in HEK293T cells that had been treated for 1 h with 30 M menadione (Fig. 7A) followed by tryptic digestion and  LC-MS/MS analysis. This analysis was repeated twice with independently prepared extracts, and extracts from non-treated cells were used as controls. In control experiments, we predominantly observed monoubiquitinated forms of pol. In these extracts, we identified seven ubiquitinated lysines (Lys 87 , Lys 271 , Lys 283 , Lys 309 , Lys 486 , Lys 488 , and Lys 508 ). All of them except Lys 508 were known as potential ubiquitination sites from previous approaches. Interestingly, in extracts prepared from menadione-treated cells where intensive polyubiquitination of pol was observed, we identified four ubiquitinated lysines (Lys 271 , Lys 309 , Lys 320 , and Lys 488 ) located in the N-terminal and central parts of pol. Three of them, Lys 271 , Lys 309 , and Lys 488 , were identified in both independent experiments. Because all four residues were previously indicated in our earlier experiments or in proteome-wide experiments (40,42,43,46,47) as potential ubiquitination sites in untreated cells, we conclude that menadione treatment most probably causes polyubiquitination of pol at lysine residues that are already monoubiquitinated rather than de novo at novel lysines.
In polyubiquitin chains, ubiquitins are linked to each other via an isopeptide bond between the C-terminal glycine of one ubiquitin and one of the lysine residues of the next ubiquitin. Ubiquitin contains seven lysine residues (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 , and Lys 63 ), and all of them can become ubiquitinated to establish polyubiquitin chains of different shape and biological function (for a review, see Ref. 36). Mass spectrometry analysis of polyubiquitinated pol revealed that the polyubiquitin chains formed in response to menadione are formed via Lys 11 and Lys 48 linkages (Fig. 9, B and C). Aside from these two peptides carrying a diglycine remnant, we also observed unmodified tryptic peptides derived from ubiquitin, including TLSDYNIQK (amino acid residues 55-63) and TITLEVEPSDTIENVK (amino acids residues 12-27). Moreover, we observed Lys 11 and Lys 48 linkages in polyubiquitinated pol obtained from plumbagin-treated cells. 3 Presumably, these linkages are formed in response to a common signal induced by exposure to naphthoquinones.
Lys 48 -linked polyubiquitin chains represent one of the most known and abundant ubiquitin linkages in the cell and target marked proteins to degradation by the 26S proteasome (60,61). The cellular role of the Lys 11 linkage is less known; however, the function of homogenous Lys 11 -linked polyubiquitin chains is also implicated in proteasomal degradation (62,63). Consistent with the notion that polyubiquitinated pol is subject to proteasomal degradation, we observed an increase in the background levels of polyubiquitinated pol in undamaged cells in the presence of the proteasome inhibitor MG132 (Fig. 10).

Discussion
Ubiquitination is an important factor allowing for quick, controlled, and reversible modification of the fate, cellular abundance, function, and localization of a protein and the promotion of protein-protein interactions. Several proteins utilized in TLS are known to be ubiquitinated in vivo, and the protein modification is used to adjust the specificity of TLS mechanisms in a variety of ways (for a review, see Ref. 6).
It has been a decade since the discovery that human pol can be monoubiquitinated in vivo (11), but the consequences of the modification remains enigmatic. Our previous studies, which show the dependence of a pol-pol interaction on the ubiquitination of either protein (13), provided some early insights into a possible role of pol modification.
In the present study, we have identified a number of lysine residues in pol that can be covalently linked to ubiquitin. Unlike pol, which is ubiquitinated at one primary site and a handful of secondary sites (6,12), we discovered that pol is ubiquitinated at more than 27 unique sites (Fig. 4). Two-thirds of the identified sites were detected in multiple autonomous studies using different experimental strategies (e.g. ectopic expression of N-and C-terminal FLAG-tagged pol versus native untagged chromosomally expressed pol and different methods of identification). Although we cannot exclude the possibility that pol is ubiquitinated at random sites, we believe that the detection of specific ubiquitination sites in multiple independent studies increases the probability that those sites are likely to play key roles in regulating the cellular activities of pol.
When assayed by SDS-PAGE, the predominant form of pol is a single monoubiquitinated species rather than multiply monoubiquitinated forms of pol. We note that under certain conditions, such as when cells are exposed to menadione, we did observe a "laddering" of FLAG-tagged pol, which is indicative of multiple monoubiquitination events (Fig. 7), but we cannot distinguish between the possibility of multiple monoubiquitinations of pol or a single monoubiquitination event that is subsequently converted into a polyubiquitin chain. Our observations therefore indicate that once pol is monoubiquitinated at one particular site it subsequently precludes monoubiquitination at additional sites in pol. Clearly, this is an area of research that needs to be studied in detail and will be the subject of future studies.
No single Lys 3 Ala or Lys 3 Arg substitution completely blocked monoubiquitination of pol. However a Lys 3 Arg substitution at Lys 715 , which is located at the very C terminus of pol and which has been shown to be ubiquitinated in two independent proteome-wide approaches (43,49), gave the greatest reduction in monoubiquitination (Fig. 3). We predict that the structure of ubiquitin covalently linked to Lys 715 will position the ubiquitin moiety for a productive interaction with the UBM2 of pol (Fig. 11E). Similarly, ubiquitination at Lys 522 may also facilitate an interaction between ubiquitin and UBM1 of pol (Fig. 11D). We hypothesize that such interactions may, in turn, help promote an interaction between pol and pol (13).
In contrast, monoubiquitination of other lysine residues may have a detrimental effect on the cellular functions of pol. For example, many sites are located in the catalytic domain of the polymerase and may alter both DNA binding properties and polymerase activity of pol (Figs. 4 and 11A and Table 2). Ubiquitination sites were also identified in both the PCNA-interacting peptide motif box and the Rev1-interacting region motif, and it seems highly unlikely that pol would be able to physically interact with either PCNA or Rev1 if these sites are ubiquitinated (Fig. 11, B and C).
Unlike pol, which is deubiquitinated upon UV irradiation (12), the level of pol monoubiquitination remained constant after exposure to a variety of DNA-damaging agents, including UV light, ethyl methanesulfonate, mitomycin C, or the oxidative DNA damage inducers potassium bromate (Fig. 5), rotenone and antimycin A (Fig. 6), and DMNQ (Fig. 8). In dramatic contrast, menadione and several structurally related naphthoquinones resulted in the rapid polyubiquitination of pol. Mass spectrometry of polyubiquitinated pol purified from menadione-and plumbagin-treated cells indicated that the polyubiquitin chains were formed through Lys 11 and Lys 48 linkages. Conjugation of ubiquitin via Lys 48 linkage is well known to serve as a signal for proteasomal degradation (60,61). Indeed, the disappearance of pol 3-5 h after exposure to menadione (Fig. 5E) is consistent with its degradation. FIGURE 10. Effect of the proteasomal inhibitor MG132 on pol ubiquitination in HEK293T cells. Cells were either untreated or exposed to the proteasomal inhibitor MG132 for the times indicated. Pol was visualized in Western blots using monoclonal antibodies to the N-terminal FLAG epitope. In both cases, the major band is unmodified pol followed by monoubiquitinated pol. We note that the intensity of polyubiquitinated pol increases over time in the MG132-treated cells compared with untreated cells, suggesting that the basal level of polyubiquitinated pol is normally kept to a minimum by 26S proteasomal degradation. Ub, ubiquitin.
We initially considered that the signal triggering polyubiquitination of pol might be oxidative DNA damage, but this was rapidly excluded when we failed to observe polyubiquitination in response to potassium bromate (Fig. 5C), DMNQ (Fig. 8), rotenone, or antimycin A (Fig. 6). However, in addition to the induction of reactive oxygen species, the naphthoquinones are FIGURE 11. Diagram of ubiquitinated lysine residues in the three-dimensional structures of human pol. A, the catalytic domain of human pol (amino acids 1-420) is shown in a ternary complex with DNA (purple tube and ladder) and an incoming dGTP (Protein Data Bank code 3GV8) (29). The pol regions are always shown as a yellow schematic with the lysine located near the functional interface highlighted as a dark blue stick. The lysines that are distal from a functional surface are shown in light blue. Lys 320 , whose ubiquitination may destabilize the protein structure, is shown in orange. Some lysine side chains are disordered and are thus modeled as alanine (Lys 51 , Lys 83 , Lys 283 , and Lys 310 ). B, the PCNA-interacting peptide motif (PIP) region of human pol. Lys 421 is near the interface with the green subunit of the trimeric PCNA (the other two PCNA subunits are shown in pink and cyan behind the green subunit) according to the crystal structure (Protein Data Bank code 2ZVM) (32). C, the Rev1-interacting region (RIR) of human pol is modeled after the crystal structures of human and mouse pol in a complex with REV1/3/7 (Protein Data Bank codes 4GK5 and 4FJO, respectively). Two lysine residues of the Rev1-interacting region (Lys 549 -Lys 550 ) are conserved, but only one (Lys 550 ) is near the interface with REV3 and forms a salt bridge with a conserved glutamate of REV1 (Glu 1174 in human REV1). D, the UBM1 of human pol is modeled after the NMR structure of mouse UBM1 in a complex with ubiquitin (Ub) (Protein Data Bank code 2KWV) (69). Lys 522 is hydrogen-bonded with the main chain carbonyl oxygen of Leu 71 in ubiquitin. E, UBM2 of human pol in complex with ubiquitin is shown according to the NMR structure (Protein Data Bank code 2L0F) (70). Lys 715 , the C-terminal residue of pol, is near the interface with ubiquitin, whereas Lys 697 and Lys 704 are distal from ubiquitin.

TABLE 2
Location of ubiquitination sites in pol and their structural implications also known to exert a wide range of cellular effects leading to stress signaling, antiangiogenesis, and thiolate arylation of proteins and amines (64 -66). One property of interest is their ability to inhibit the lysine acetyltransferase (KAT) p300 (58,59). All of the naphthoquinones (1,4-naphthoquinone, menadione, juglone, and plumbagin) that induced pol polyubiquitination have previously been reported to inhibit the KAT activity of p300 (59). It is unknown whether DMNQ (which did not cause pol polyubiquitination) can inhibit the KAT activity of p300. However, because DMNQ lacks the critical hydroxyl group at the fifth position of the benzene ring that is required for inhibition of KAT activity (59) and the second and third positions that are normally subject to nucleophilic attack are occupied by methoxy groups (Fig. 8), we assume that it probably does not inhibit KAT activity.
It is now well established that acetylation is a key regulator of diverse biological processes from metabolism to signaling and immunity (67). Indeed, like many proteins, ubiquitin is subject to acetylation (49,68). Interestingly, both Lys 11 and Lys 48 are moderately sensitive to acetylation (49,68). Thus, we hypothesize that if Lys 11 and Lys 48 of ubiquitin are acetylated it would preclude the formation of polyubiquitin chains via these linkages. Our observation that the p300 inhibitor L002 also induces polyubiquitination of pol strongly suggests that there is a competition between ubiquitination and acetylation at overlapping lysine residues in pol. We believe that such competition constitutes a novel mechanism to regulate the stability of pol that warrants further investigation.